Low G+C Gram-positive bacteria possess the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which transports and initiates the metabolism of its sugar substrates and influences the processes of catabolite repression (CR) and catabolite activation (CA). We and others have shown that a metabolite-activated, ATP-dependent protein kinase, PtsK, at least in part mediates regulation in Bacillus subtilis, our model organism, by phosphorylating seryl residue-46 in the paralogous phosphocarrier proteins of the PTS, HPr (the major co-repressor present in all low G+C Gram-positive bacteria) and Crh (the minor co-repressor present only in Bacilli). These phosphorylated PTS proteins interact directly with at least one well-characterized transcription factor, CcpA, to influence gene expression. We have discovered a second transcription factor, CcpB, which mediates catabolite repression under certain growth conditions and have shown that it exerts pleiotropic effects on gene expression. However, the mechanism of its action is largely unknown. Further, our transcriptome results showed that CcpA regulates many genes following patterns that cannot be explained on the basis of the established mechanism involving PtsK. We have cloned, sequenced and put into expression vectors all of the known genes encoding the constituents of these regulatory pathways. In the proposed research, we will combine transcriptome, bioinformatic, molecular genetic and biochemical approaches to establish the molecular mechanisms and physiological significance of CR and CA in B. subtilis. To identify targets and signal transduction pathways of CR and CA, we will (a) conduct whole transcriptome analyses of wild-type B. subtilis and a complete complement of isogenic mutants defective or altered for CR-mediating genes: ccpA, ccpB, ptsK, ptsH1 and crh, (b) use bioinformatic approaches to define the DNA binding sites that promote CcpA and CcpB-mediated regulation, (c) use reporter gene fusions and molecular genetic approaches in vivo to confirm and extend the results obtained in (a) and (b), and (d) use in vitro biochemical approaches to establish the detailed molecular mechanisms of regulation. This research will reveal the molecular details and physiological consequences of protein kinase-mediated and protein kinase-independent CR and CA in low G+C Gram-positive prokaryotes, including a broad range of human pathogens.